Understanding the mechanisms of an effective neutralizing antibody response to HIV is one of the highest priorities in the field of HIV-specific immunity. In this regard, the inability of the humoral response of most vaccinees to cross-neutralize multiple strains of HIV is believed to be a major obstacle to the design of effective vaccines. In 2006 we observed that sera from subpopulation of our chronically infected cohorts had considerable neutralization breadth extending across clades. Much of the work done prior to that time had not focused on patients selected for broadly cross-neutralizing antibodies to HIV-1. In addition, a relatively small number of monoclonal neutralizing antibodies existed at the time. For these reasons we recruited a cohort of individuals screened for broadly cross-neutralizing antibodies to HIV. We, in collaboration with investigators at the Vaccine Research Center (VRC), used these sera to systematically dissect the means by which these patients cross-neutralize HIV-1. We thus far have identified 30 such patients and are continuing to accrue additional subjects. A number of fundamental questions had not been addressed with regard to the HIV-specific humoral immune response of these patients. For example, it was not known if these patients had genetic or clinical characteristics, or HIV-specific cellular immune response characteristics in common. Given that our patients are infected with clade B viruses and should not have experienced infection by viruses belonging to multiple clades, we hypothesized that cross-neutralization is mediated through conserved epitopes on HIV envelope (Env). In addition, although considerable work had been done on patient sera, very little had been done on HIV-specific B cells. The phenotype and immunoglobulin class of HIV-specific B cells in comparison to responses to other viruses remained poorly defined. Further, it remained unclear whether patients with broad cross-neutralizing activity are unique with regard to these parameters. One primary objective of our work on the humoral response to HIV is to understand the basis of a broadly cross-neutralizing antibody response in our patients. It was not known whether there are common features of the humoral response of such patients with regard to specificity. It was also not known whether neutralization was mediated by a few B cell clones directed to conserved epitopes or by an extremely polyclonal response to many epitopes. To dissect the specificity and diversity of epitopes targeted by the B-cell response in patients with broad sera, we have initiated a collaborative effort to isolate monoclonal antibodies. To further understand the specificities and binding characteristics that underlie a broadly neutralizing antibody response we developed techniques that permitted isolation of human monoclonal antibodies without previous knowledge of specificity. In this technique peripheral blood memory B cells are sorted and expanded for 13 days with interleukin (IL)-2, IL-21 and CD40-ligand expressing cells. The supernatants of large numbers of micro-cultures of these cells can then be screened for neutralizing activity in a high-throughput manner. From the cultures that exhibit anti-HIV neutralizing activity the immunoglobulin genes can then be isolated, re-expressed, and characterized. In the recent past we have used these techniques to isolate antibodies with novel specificities on HIV Env that represent conserved sites of vulnerability that can be targeted in prophylaxis and immunotherapies. We previously isolated an antibody, designated 10E8, which is among the most broad thus far described. It binds the gp41 membrane-proximal external region (MPER) of Env. It neutralizes 98% of tested viruses. 10E8 also provided protection from mucosal challenge with a simian immunodeficiency virus expressing an HIV Env glycoprotein. We have also isolated an antibody termed 35O22 that neutralizes by binding a conserved face on contiguous areas of gp41 and gp120. This was one of a novel class of antibodies that bind across the gp41-120 interface. More recently, we have isolated a CD4-binding site antibody, termed N6, that potently neutralized 98% of HIV-1 isolates, including 16 of 20 that were resistant to other members of its class. These results suggested that such broadly neutralizing antibodies provide a rationale for use of these antibodies in prophylaxis or induction of these antibodies by vaccines to prevent HIV infection. These antibodies are among the lead candidates for clinical development by research and corporate collaborators. Through collaborative work N6 and 10E8 have been further modified to either increase potency and manufacturability. Some collaborators have incorporated N6 or 10E8 sequences into bi- or tri-specific antibodies that increase the breadth or potency compared to individual antibodies. Passive administration of single, bi-, or tri-specific antibodies are in various stages of pre-clinical or clinical development for therapy or prophylaxis. In parallel we have pursued a better understanding of the features of a vaccine platform that might result in the induction of broad and durable antibody responses to HIV. Of the available vaccine platforms for viral surface glycoproteins, replicating vectors have the potential to be highly immunogenic and offer several advantages over most non-replicating vectors. They can express viral surface glycoproteins such that the total dose and duration of exposure to antigen likely exceed those of non-replicating vectors. In addition, viral surface glycoproteins may be expressed by the host cell in the appropriate conformation and glycosylation state. They may therefore more closely match those expressed during natural infection compared to vaccines produced in heterologous cell lines or eggs. In addition, replicating vectors can cause inflammation through the induction of pro-inflammatory cytokines. They may also directly or indirectly stimulate B cell proliferation and differentiation through nucleic acid stimulation of toll-like receptors within B cells or antigen presenting cells such as follicular dendritic cells. Although replicating vectors offer numerous advantages, the level of immunogenicity is not assured. It is potentially modulated by the replicative capacity of the vector itself, transgene expression, pre-existing immunity, and route of administration. We began this work using a recombinant replication-competent Adenovirus type 4 that encoded an influenza virus hemagglutinin type 5 Vietnam (Ad4-H5-Vtn). This adenovirus was chosen given its extraordinary safety record and its ability to replicate in humans for 2-4 weeks. H5 was used as a model antigen, that permitted the study of the dose, route, and transmissibility prior to the study of HIV recombinants. The recombinant Ad4-H5-Vtn was given as an enteric coated capsule, or applied to the tonsils with a swab, or given as an intranasal spray. Surprisingly, among participants that received the tonsillar or intranasal vaccine, we observed evolution of the B cell response specificity and antibody affinity maturation 6-12 months beyond the period of active viral replication. Although serum neutralization after a single infection was modest, individual antibodies that were broad and potent could be isolated including one stem-specific antibody that shares genetic elements and a mode of recognition with a previously described antibody, named 39.29, thus forming a new multi-donor class of broad antibodies for influenza virus. These results suggested that evolution of B cell specificity and antibody affinity maturation can persist for months following a single vaccination. They further suggest that the replication-competent Ad4 platform shows considerable promise for the induction of a durable, broad antibody response to